Interferon-Gamma (IFN-γ)
Interferons are relatively small, single-chain glycoproteins released by cells invaded by viruses or certain other substances. Interferons are presently grouped into three major classes, designated leukocyte interferon (interferon-alpha, α-interferon, IFN-α), fibroblast interferon (interferon-beta, β-interferon, IFN-β), and immune interferon (interferon-gamma, γ-interferon, IFN-γ). In response to viral infection, lymphocytes synthesize primarily α-interferon (along with a lesser amount of a distinct interferon species, commonly referred to as omega interferon), while infection of fibroblasts usually induces β-interferon. α- and β-interferons share about 20–30 percent amino acid sequence homology. The gene for human IFN-β lacks introns, and encodes a protein possessing 29% amino acid sequence identity with human IFN-αI, suggesting that IFN-α and IFN-β genes have evolved from a common ancestor (Taniguchi et al., Nature 285, 547–549 [1980]). By contrast, IFN-γ is not induced by viral infection, rather, is synthesized by lymphocytes in response to mitogens, and is scarcely related to the other two types of interferons in amino acid sequence. Interferons-α and -β are known to induce MHC Class I antigens, while IFN-γ induces MHC Class II antigen expression, and also increases the efficiency with which target cells present viral peptide in association with MHC Class I molecules for recognition by cytotoxic T cells.
IFN-γ is a member of the interferon family, which exhibits the antiviral and anti-proliferative properties characteristic of interferons-α and -β (IFN-α and IFN-β) but, in contrast to those interferons, is PH 2 labile. IFN-γ was originally produced upon mitogenic induction of lymphocytes. The recombinant production of human IFN-γ was first reported by Gray, Goeddel and co-workers (Gray et al., Nature 295, 503–508 [1982]), and is subject of U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138, 4,925,793, 4,855,238, 5,582,824, 5,096,705, 5,574,137, and 5,595,888. The recombinant human IFN-γ of Gray and Goeddel as produced in E. coli, consisted of 146 amino acids, the N-terminal position of the molecule commencing with the sequence CysTyrCys. It has later been found that the native human IFN-γ (i.e., that arising from mitogen induction of human peripheral blood lymphocytes and subsequent purification) is a polypeptide which lacks the CysTyrCys N-terminus assigned by Gray et al., supra. More recently, the crystal structure of E. coli derived recombinant human IFN-γ (rhIFN-γ) was determined (Ealick et al., Science 252, 698–702 [1991]), showing that the protein exists as a tightly intertwined non-covalent homodimer, in which the two identical polypeptide chains are oriented in an antiparallel manner.
IFN-γ is known to exhibit a broad range of biological activities, including antitumor, antimicrobial and immunoregulatory activities. A particular form of recombinant human IFN-γ (rhIFN-γ-1b, Actimmune®, Genentech, Inc. South San Francisco, Calif.) is commercially available as an immunomodulatory drug for the treatment of chronic granulomatous disease characterized by severe, recurrent infections of the skin, lymph nodes, liver, lungs, and bones due to phagocyte disfunction (Baehner, R. L., Pediatric Pathol. 10, 143–153 [1990]). IFN-γ has also been proposed for the treatment of atopic dermatitis, a common inflammatory skin disease characterized by severe pruritus, a chronically relapsing course with frequent periods of exacerbation, a distinctive clinical morphology and distribution of skin lesions (see PCT Publication No. WO 91/07984 published 13 Jun. 1991), vascular stenosis, including the treatment of restenosis following angioplasty and/or vascular surgery (PCT Publication No. WO 90/03189 published 5 Apr. 1990), various lung conditions, including respiratory distress syndromes (RDS), such as adult respiratory distress syndrome (ARDS) and a neonatal form, termed variously as idiopathic RDS or hyaline membrane disease (PCT Publication No. WO 89/01341, published 23 Feb. 1989). In addition, IFN-γ has been proposed for use in the treatment of various allergies, e.g. asthma, and HIV-infection-related conditions, such as opportunistic infections, e.g. Pneumocystis carinii pneumonia, and trauma-associated sepsis. Impaired IFN-γ production has been observed in multiple-sclerosis (MP) patients, and it has been reported that the production of IFN-γ is greatly suppressed in suspensions of mitogen-stimulated mononuclear cells derived from AIDS patients. For a review see, for example, Chapter 16, “The Presence of Possible Pathogenic Role of Interferons in Disease”, In: Interferons and other Regulatory Cytokines, Edward de Maeyer (1988, John Wilet and Sons Publishers).
Interferon-γ, along with other cytokines, has been implicated as an inducer of inducible nitric oxide (iNOS) which, in turn, has been described as an important mediator of the inflammatory mechanism underlying heart failure, of the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies. Ungureanu-Longrois et al., Circ. Res. 77, 494–502 (1995); Pinsky et al., J. Clin. Invest. 95, 677–685 (1995); Singh et al., J. Biol. Chem. 270, 28471–8 (1995); Birks and Yacoub, Coronary Artery Disease 8, 389–402 (1997); Hattori et al., J. Mol. Cell. Cardiol. 29, 1585–92 (1,997). Indeed, IFN-γ has been reported to be the most potent single cytokine with regard to myocyte iNOS induction (Watkins et al., J. Mol. & Cell. Cardiol. 27, 2015–29 [1995]).
Cardiac Hypertrophy
Hypertrophy is generally defined as an increase in size of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both.
Cardiac hypertrophy is the enlargement of heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output or to injury. Morgan and Baker, Circulation 83, 13–25 (1991). This response is frequently associated with a variety of distinct pathological conditions, such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, cardiac shunt, congestive heart failure, etc.
On a cellular level, the heart functions as a syncytium of myocytes and surrounding support cells, called non-myocytes. While non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothelial and smooth muscle cells. Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart.
The enlargement of embryonic heart is largely dependent on an increase in myocyte number, which continues until shortly after birth, when cardiac myocytes lose their proliferative capacity. Further growth occurs through hypertrophy of the individual cells. Hypertrophy of adult cardiac ventricular myocytes is a response to a variety of conditions which lead to chronic hemodynamic overload. Thus, in response to hormonal, physiological, hemodynamic, and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process. This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atrial natriuretic peptide (ANP). Chien et al., FASEB J. 5, 3037–3046(1991); Chien et al., Annu. Rev. Physiol. 55, 77–95 (1993). An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around intramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure overload in humans (Caspari et al., Cardiovasc. Res. 11, 554–8 [1977]; Schwarz et al., Am. J. Cardiol. 42, 895–903 [1978]; Hess et al., Circulation 63, 360–371 [1981]; Pearlman et al., Lab. Invest. 46, 158–164 [1982]). Cardiac hypertrophy due to chronic hemodynamic overload is the common end result of most cardiac disorders and a consistent feature of cardiac failure.
It has also been suggested that paracrine factors produced by non-myocyte supporting cells may additionally be involved in the development of cardiac hypertrophy, and various non-myocyte derived hypertrophic factors, such as, leukocyte inhibitory factor (LIF) and endothelin, have been identified. Metcalf, Growth Factors 7, 169–173 (1992); Kurzrock et al., Endocrine Reviews 12, 208–217 (1991); Inoue et al., Proc. Natl. Acad. Sci. USA 86: 2863–2867 (1989); Yanagisawa and Masaki, Trends Pharm. Sci. 10, 374–378 (1989); U.S. Pat. No. 5,573,762 (issued Nov. 12, 1996). Further exemplary factors that have been identified as potential mediators of cardiac hypertrophy include cardiotrophin-1 (CT-1) (Pennica et al., Proc. Nat. Acad. Sci. USA 92:1142–1146 [1995]), catecholamines, adrenocorticosteroids, angiotensin, and prostaglandins.
Adult myocyte hypertrophy is initially beneficial as a short term response to impaired cardiac function by permitting a decrease in the load on individual muscle fibers. With severe, long-standing overload, however, the hypertrophied cells begin to deteriorate and die. Katz, “Heart Failure”, in: Katz A. M. ed., Physiology of the Heart (New York, Raven Press, 1992) pp. 638–668. Cardiac hypertrophy is a significant risk factor for both mortality and morbidity in the clinical course of heart failure. Katz, Trends Cardiovasc. Med. 5, 37–44 (1995).
For further details of the causes and pathology of cardiac hypertrophy see, e.g. Heart Disease, A Textbook of Cardiovascular Medicine, Braunwald, E. ed., W.B. Saunders Co., 1988, Chapter 14, Pathophysiology of Heart Failure.
Treatment of Cardiac Hypertrophy
At present, the treatment of cardiac hypertrophy varies depending on the underlying cardiac disease. Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, leukemia inhibitory factor (LIF), endothelin (including endothelin-1, -2, and -3 and big endothelin), cardiotrophin-1 (CT-1) and cardiac hypertrophy factor (CHF) are among the factors identified as potential mediators of hypertrophy.
For example, β-adrenergic receptor blocking drugs (β-blockers, e.g., propranolol, timolol, tertalolol, carteolol, nadolol, betaxolol, penbutolol, acetobutolol, atenolol, metoprolol, carvedilol, etc.) and verapamil have been used extensively in the treatment of hypertrophic cardiomyopathy. The beneficial effects of β-blockers on symptoms (e.g. chest pain) and exercise tolerance are largely due to a decrease in the heart rate with a consequent prolongation of diastole and increased passive ventricular filling. Thompson et al., Br. Heart J. 44, 488–98 (1980); Harrison et al., Circulation 29, 84–98 (1964). Verapamil has been described to improve ventricular filling and probably reducing myocardial ischemia. Bonow et al., Circulation 72, 853–64 (1985). Nifedipine and diltiazem have also been used occasionally in the treatment of hypertrophic cardiomyopathy. Lorell et al., Circulation 65, 499–507 (1982); Betocchi et al., Am. J. Cardiol. 78, 451–7 (1996). However, because of its potent vasodilating properties, nifedipine may be harmful, especially in patients with outflow obstruction. Disopyramide has been used to relieve symptoms by virtue of its negative inotropic properties. Pollick, N. Engl. J. Med. 307, 997–9 (1982). In many patients, however, the initial benefits decrease with time. Wigle et al., Circulation 92, 1680–92 (1995).
Antihypertensive drug therapy has been reported to have beneficial effects on cardiac hypertrophy associated with elevated blood pressure. Examples of drugs used in antihypertensive therapy, alone or in combination, are calcium antagonists, e.g. nitrendipine; β-adrenergic receptor blocking agents, e.g., those listed above; angiotensin converting enzyme (ACE) inhibitors, e.g., quinapril, captopril, enalapril, ramipril, benazepril, fosinopril, lisinopril; diuretics, e.g. chorothiazide, hydrochlorothiazide, hydroflumethazide, methylchlothiazide, benzthiazide, dichlorphenamide, acetazolamide, indapamide; calcium channel blockers, e.g. diltiazem, nifedipine, verapamil, nicardipine. For example, treatment of hypertension with diltiazem and captopril showed a decrease in left ventricular muscle mass, but the Doppler indices of diastolic function did not normalize. Szlachcic et al., Am. J. Cardiol. 63, 198–201 (1989); Shahi et al., Lancet 336, 458–61 (1990). These findings were interpreted to indicate that excessive amounts of interstitial collagen may remain after regression of left ventricular hypertrophy. Rossi et al., Am. Heart J. 124, 700–709 (1992). Rossi et al., supra, investigated the effect of captopril on the prevention and regression of myocardial cell hypertrophy and interstitial fibrosis in pressure overload cardiac hypertrophy, in experimental rats.
As there is no generally applicable therapy for the treatment of cardiac hypertrophy, the identification of factors that can prevent or reduce cardiac myocyte hypertrophy is of primary importance in the development of new therapeutic strategies to inhibit pathophysiological cardiac growth.